Robert L. Wolfert

Nonpalpable stage t1c prostate cancer : Prediction of insignificant disease using free/total prostate specific antigen levels and needle biopsy findings

PURPOSE Approximately 25% of radical prostatectomies performed for stage T1c disease show potentially insignificant prostate cancer. We previously reported the use of serum prostate specific antigen (PSA) density and needle biopsy findings to predict potentially insignificant cancer. We now evaluate whether using free/total serum PSA levels along with needle biopsy findings can better predict tumor significance. MATERIALS AND METHODS We studied 163 radical prostatectomy specimens of stage T1c prostate cancer in which free/total serum PSA levels were determined. Free/total serum PSA levels were measured with Tandem-MP PSA assays. Insignificant prostate cancers were organ confined with tumor volumes less than 0.5 cc and Gleason score less than 7. Advanced tumors were either Gleason score 7 or greater, established extraprostatic extension with positive margins, or positive seminal vesicles or lymph nodes. Other cases were considered as moderate tumor. Moderate and advanced tumors were considered significant. RESULTS Of the tumors 30.7% were insignificant, 49.7% moderate and 19.6% advanced. The best model to predict preoperatively insignificant tumor was a free/total PSA of 0.15 or greater and favorable needle biopsy findings (less than 3 cores involved, none of the cores with greater than 50% tumor involvement and Gleason score less than 7). Using this model of the 18 tumors predicted to be insignificant 17 were insignificant for a positive predictive value of 94.4%. Of the 145 cases that were predicted to be significant 112 were correctly predicted for a negative predictive value of 77.2%. There was only 1 tumor predicted to be insignificant which was classified as moderate. No tumor predicted to be insignificant was advanced. CONCLUSIONS In conjunction with needle biopsy findings, free/total PSA levels accurately predict insignificant tumor in stage T1c disease.

A precursor form of PSA (pPSA) is a component of the free PSA in prostate cancer serum

OBJECTIVES Prostate-specific antigen (PSA) is a widely used serum marker for human prostate cancer (PCa). The majority of PSA in serum is present as a complex with alpha-1-antichymotrypsin (ACT). In recent years, the ratio of free (uncomplexed) to total PSA has shown improved discrimination of PCa from benign prostatic hyperplasia. This study examines the nature of the free PSA from detected in PCa serum and shows that some of the uncomplexed PSA is an inactive precursor of PSA (pPSA). METHODS Western blot analysis was used to detect clipped, fragment forms of PSA in sera and seminal fluid. Hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC) was used to identify forms of PSA present in the free PSA population. Pooled sera was passed over a PSA immunoaffinity column, and the eluted PSA components were further resolved by HIC-HPLC. RESULTS Western blot analysis of whole sera showed complexed PSA and the intact, approximately 34 kilodalton free PSA. Only negligible levels of clipped or degraded forms of PSA, as found in seminal fluid, were detected. Column fractions measured for uncomplexed PSA using the Tandem-MP free PSA assay showed that about 25% of the free PSA eluted as pPSA beginning at the [-4]amino acid. Studies with purified recombinant [-4]pPSA showed that this proenzyme form is inactive and does not complex with ACT. CONCLUSIONS These results suggest that the uncomplexed PSA in PCa serum is primarily unclipped PSA that contains a significant fraction of pPSA.

Use of human glandular kallikrein 2 for the detection of prostate cancer: Preliminary analysis

OBJECTIVES Human glandular kallikrein 2 (hK2) and prostate-specific antigen (PSA) are members of a multigene family of serine proteases that share approximately 80% sequence homology. Both are expressed in the prostate epithelium, are under androgen regulation, are present in serum and seminal fluid, and can form complexes with endogenous protease inhibitors (eg, alpha2-macroglobulin and alpha1-antichymotrypsin). Differences in immunohistochemistry and substrate specificity suggest hK2 may provide unique information for early detection and characterization of prostate cancer. METHODS Nine hundred thirty-seven archived serum samples from men treated at two academic institutions were studied. All men underwent biopsy, had a histologically confirmed diagnosis of cancer or noncancer, and a total PSA level greater than 2 ng/mL. Samples were tested in Hybritechs Tandem-R PSA and Tandem-R free PSA (fPSA) assays and a research prototype assay for total hK2 (thK2). RESULTS The thK2/fPSA ratio provided additional specificity for cancer detection over PSA and the percentage of fPSA (%fPSA). A model for cancer detection using %fPSA and the thK2/fPSA ratio when PSA is 2 to 4 ng/mL is proposed that would identify as many as 40% of the cancers and would require biopsy in only 16.5% of the men in this PSA range. CONCLUSIONS In this study, %fPSA and thK2/fPSA provided unique information for prostate cancer detection and increased the specificity of cancer detection.

Human glandular kallikrein 2 expression in prostate adenocarcinoma and lymph node metastases

OBJECTIVES To describe the expression of a potential new tumor marker, human glandular kallikrein 2 (hK2), in primary adenocarcinoma and lymph node metastases that may be useful as an adjunct to prostate-specific antigen (PSA) in the diagnosis and monitoring of prostate cancer. METHODS We evaluated 151 radical prostatectomy specimens removed at Mayo Clinic with node-positive adenocarcinoma to compare cytoplasmic expression of hK2, pro-hK2, and PSA in benign tissue, prostate adenocarcinoma, and lymph node metastases. Monoclonal antibodies for mature hK2 (hK2-G586), pro-hK2 (pro-hK2-G464), and PSA (PSA-773) were used. A polyclonal antibody for PSA was also used. Immunoreactivity in each case was tested to determine whether cancer recurrence could be predicted. RESULTS Intense epithelial cytoplasmic immunoreactivity was observed in every case for hK2-G586, pro-hK2-G464, PSA-773, and polyclonal PSA (100% of cases, respectively). The intensity and extent of hK2 expression was greater in lymph node metastases than in primary cancer; furthermore, the expression in primary cancer was greater than in benign epithelium. Pro-hK2 was expressed in a greater percentage of cells in primary cancer than in benign tissue; furthermore, pro-hK2 was expressed to a greater extent in primary cancer than in lymph node metastases. In marked contrast to mature hK2, monoclonal PSA immunoreactivity was expressed to a higher extent in primary cancer than in lymph node metastases. Polyclonal PSA showed an incremental increase in expression from benign tissue to primary cancer and a further increase in expression in lymph node metastases. CONCLUSIONS hK2 was expressed in every cancer, and the expression incrementally increased from benign epithelium to primary cancer and lymph node metastases. Pro-hK2 was expressed to the greatest extent in primary cancer. Monoclonal PSA displayed inverse immunoreactivity compared with hK2. Polyclonal PSA showed incremental increases, suggesting that both hK2 and PSA were being detected. Tissue expression of hK2 appears to be regulated independently of PSA in benign epithelium, adenocarcinoma, and lymph node metastases.

Use of percentage of free prostate-specific antigen to identify men at high risk of prostate cancer when psa levels are 2.51 to 4 ng/mL and digital rectal examination is not suspicious for prostate cancer: an alternative model

OBJECTIVES Currently, many clinicians do not recommend prostate biopsy for men with digital rectal examination (DRE) results that are not suspicious for cancer and prostate-specific antigen (PSA) values between 2.51 and 4 ng/mL. We propose a new model for the detection of prostate cancer using the percentage of free PSA (%FPSA) in the limited range of PSA values between 2.51 and 4 ng/mL that maximizes clinical specificity (ie, minimizes false-positive results). This model identifies higher risk patients in this relatively low-risk population. METHODS Three hundred sixty-eight archived serum samples from men evaluated and treated at two academic institutions were reviewed. All men had a histologic diagnosis, findings not suspicious for cancer on DRE, and PSA levels between 2.51 and 4 ng/mL. Samples were tested in Hybritechs Tandem-R PSA and Tandem-R free PSA (FPSA) assays in the same laboratory at each institution. RESULTS Various models for cancer detection using %FPSA when PSA is 2.51 to 4 ng/mL and DRE is not suspicious for cancer are proposed. These models recommend biopsy for only 10% to 36% of the men in this population and would identify as many as 30% to 54% of the detectable cancers. There is evidence that the cancers that would be detected are the most aggressive cancers in this population. CONCLUSIONS Our models identified men with a higher risk of prostate cancer in a relatively low-risk population that currently does not routinely undergo biopsy. This may allow for a more cost-effective way to increase cancer detection when PSA values are between 2.51 and 4 ng/mL and DRE is not suspicious for cancer. This model has the potential to detect a greater number of clinically important and potentially curable cancers than would be detected with current practice.

Development of monoclonal antibodies specific for human glandular kallikrein (hK2): development of a dual antibody immunoassay for hK2 with negligible prostate-specific antigen cross-reactivity.

OBJECTIVES Human glandular kallikrein (hK2) is a protein that is 80% homologous to prostate-specific antigen (PSA), and, like PSA, is localized to the prostate. We developed a specific immunoassay for hK2 that can be used to evaluate its clinical diagnostic utility. METHODS We developed monoclonal antibodies (mAbs) specific for hK2 by immunizing with hK2 and screening for clones reactive with hK2 and not PSA. Prototype sandwich assays using these mAbs were tested, and the optimum pair selected. Purified hK2 was used as standard and PSA cross-reactivity was assessed in the assay. Both hK2 and hK2-alpha1-antichymotrypsin (ACT) complexes have been identified in sera of patients with prostate cancer (PCa). Serum samples (n = 671) from healthy volunteers and patients with prostate disease were assayed for hK2 and PSA levels. RESULTS The assay had a detection limit of less than 0.12 ng/mL and a less than 0.5% cross-reactivity with PSA. The assay preferentially detected free hK2 with a 3.5-fold higher molar response than with hK2-ACT. The mean serum concentration of hK2 in normal control samples was low (0.33 and 0.37 ng/mL for normal healthy men and women, respectively) but was elevated in patients with prostate disease (0.86 and 6.77 ng/mL for patients with benign prostatic hyperplasia and PCa, respectively). Negligible cross-reactivity to hK2 was measured by Tandem PSA assays (Hybritech). CONCLUSIONS Significant concentrations of hK2, relative to PSA, were detected in human serum, especially in patients with prostate disease. Serum hK2 concentrations were not proportional to PSA concentration. Therefore, hK2 has the potential to be an independent and clinically useful marker for PCa.

Measurement of serum prostate-specific membrane antigen, a new prognostic marker for prostate cancer

OBJECTIVES To describe current results with Western blot assay for prostate specific membrane antigen (PSMA) using 7E11.C5 antibody and the development of an additional antibody measurement for PSMA by a new sandwich immunoassay. METHODS A population of patients from a screening group, from a difficult diagnostic group, from a pre- and postoperative radical prostatectomy group, and from a group with metastatic disease followed for a serial period, provided the serum values for a prospective assessment of PSMA by Western blot assay. A new monoclonal antibody was sought, reacting to the C-terminal region of PSMA in order to develop a sandwich radioimmunoassay. RESULTS PSMA values in screened patients correlate with the more advanced stage of the cancers determined. In postprostatectomy patients, the PSMA value corresponds more with preoperative values and with the values of those with a poor clinical course. In difficult diagnostic cases, the PSMA value is increased, specifically in hormone-refractory cases and particularly in those cases judged by other criteria, such as the National Prostatic Cancer Project, to be in clinical progression compared with those judged to be in clinical remission. The level of PSMA value appears to be independent of homogeneous tumor volume and to be more related to that of prior hormone treatment, or to where prostate cancer cells can be documented to be outside the prostate. A new monoclonal antibody, 3F5.4G6, reacts with the extracellular domain of PSMA near the C-terminal region. This is in contrast to the previously measured antibody 7E11.C5, which reacts with an N-terminal epitope. 3F5.4G6 recognizes the same PSMA protein as does 7E11.C5. The epitopes are essentially at opposite ends of the molecule. The 3F5.4G6 antibody reacts with the LNCaP line but not with DU145, or PC3. These two antibodies to PSMA are well suited for use in a new sandwich immunoassay. CONCLUSIONS PSMA provides a prostatic cancer serum test by using Western blot, which suggests a clinical prognostic value not seen with other markers. New antibodies, such as 3F5.4G6, reacting with the extracellular domain of PSMA combined with 7E11.C5, appear to offer an opportunity for a new sandwich immunoassay.

Clearance rate of serum-free and total PSA following radical retropubic prostatectomy

Our objective was to determine the clearance rate of free and total serum PSA following radical retropubic prostatectomy.

Tartrate-Resistant Acid Phosphatase Forms Complexes with α2-Macroglobulin in Serum

Tartrate‐resistant acid phosphatase (TRAP) is a standard histochemical marker of differentiated osteoclasts and has been proposed as a serum/plasma marker for osteoclast activity. Enzyme assays have been described that show elevated TRAP enzyme activity in the serum or plasma of patient groups known to have increased bone metabolism. However, the poor stability of the enzyme and potential contribution from nonosteoclastic sources make it problematic to measure in patient samples. Immunoassays developed to measure TRAP in serum and plasma have yielded widely varying TRAP levels in both normal and disease states. It is not clear if this variability is caused by differences in assay calibration, antibody specificity, and/or TRAP instability. In this paper, we report that purified TRAP spiked into serum forms high molecular weight complexes. Complex formation results in greatly decreased TRAP enzyme activity and immunoreactivity. The complexing protein in serum has been identified as α2‐macroglobulin (α2M). Similar complexes are observed in stored patient samples. In vitro studies with purified components show that TRAP binds to α2M primarily through noncovalent ionic interactions. Our results demonstrate that one mechanism of TRAP instability in serum is complex formation with α2M and suggest further that current TRAP enzyme and immunoassays may not accurately measure the circulating level of TRAP.

Dual monoclonal antibody immunoassay for free prostate-specific antigen

Prostate‐specific antigen (PSA) is the most important tumor marker for early detection and monitoring of prostate cancer (PCa) patients. PSA is also elevated in many patients with benign prostatic hyperplasia (BPH). The study of the serum PSA forms, free PSA (f‐PSA) and PSA complexed with α1‐antichymotrypsin (PSA‐ACT), may improve the discrimination between PCa and BPH. An immunoassay specific for f‐PSA is reported with very low cross‐reactivity (0.7%) to PSA‐ACT. Serum specimens from BPH and PCa patients (determined by biopsy) with PSA levels from <1 to >100 ng/ml were tested. No f‐PSA was detected in serum specimens from normal females (N = 50). Low levels (0–0.3 ng/ml) were detected in specimens from healthy males (N = 60). In specimens from PCa and BPH patients, the f‐PSA to total PSA ratio (f/t) was found to range from 1% to higher than 60%. While maintaining an 80% sensitivity for cancer, the f/t ratio improved specificity to approximately 80%, as compared to 55% for total PSA alone. The receiver operating characteristics (ROC) curve analysis of the f/t ratio displayed a greater area under the plot (0.84) compared to total PSA alone (0.745). The results demonstrate that the f/t ratio significantly increases specificity for PCa detection compared to total PSA alone, showing the potential clinical value of the f‐PSA immunoassay.